Until now, no one has performed chemical preparations as disclosed herein. Devices are used that emit radiofrequency/microwave energy. The energy is directed to a target object, for example, a microarray chip or a microtiter plate, that contains one or more material(s) that absorb(s) microwave energy. The microwave-generated heat energy accelerates a desired chemical reaction on the surface of the targeted object.
Microwave Chemistry
Microwaves (including radiofrequency or RF electromagnetic radiation) are commonly used in wireless communication devices. Advances in microwave transmission have improved along with tremendous recent technological improvements in the satellite and communications industry (for example, in cell phones and wireless internet).
Microwaves are also well known in common kitchen appliances. Microwave ovens heat water-containing food rapidly because water is efficient at converting microwave energy to thermal energy. Kitchen microwave ovens emit microwaves at a frequency of 2.45 GHz, which is well within the microwave absorption spectrum of water. Frequencies outside of the absorption spectrum of water would not heat food as well.
Another use for microwaves is in chemical reaction applications (Bose et al., 1997; Bradley, 2001; Wathey et al., 2002; Lew et al., 2002). Microwave chemistry refers to the use of microwaves to accelerate chemical reactions. Reactions are usually carried out using microwave radiation to heat bulk solutions that contain the reactants (Mingos & Baghurst, 1991; Zlotorzynski, 1995). Often these reactions are carried out in non-aqueous solvents. Microwave ovens specifically designed for use in carrying out microwave chemistry of bulk reaction solutions are commercially available (CEM Corporation (Mathews, N.C.), Milestone, Inc. (Monroe, Conn.), Personal Chemistry AB (Uppsala, Sweden), PerkinElmer Instruments (Shelton, Conn.)).
Microwave accelerated reactions are sometimes run on solvent-free supports such as alumina and silica (Varma, 2001; Bose, 1997; Bram et al., 1990). The supports can be doped with reagents, for example in detoxifying waste. The supports are chosen because they are inexpensive and recyclable agents which non-specifically adsorb/extract the reagent of interest. No specific binding of (such as by antibodies) is used to capture reagents.
Microwave-enhanced catalysis has also been described (Roussy & Pearce, 1995). The term “microwave-enhanced catalysis” has been used to refer to conventional catalysis, rather than to catalysis that occurs in enzyme-like binding pockets in aqueous solution. One example of such usage of the term “microwave-enhanced catalysis” is the isomerization of liquid hexane using a metallic Pt/Al2O3 catalyst. Another example is the partial oxidation of gaseous methane using a catalyst that is an oxide of SmLiO2 doped with CaO and MgO (Roussy & Pearce, 1995).
Another example of the application of microwaves to accelerate chemical reactions is the use of microwave-absorbing particles to enhance the heating of a bulk solution (Holzwarth et al., 1998). In this case, dispersed cobalt and magnetite nanoparticles were used as microwave (2.45 GHz) absorbers to heat a bulk xylene solution. Xylene is a non-polar solvent not appreciably heated by microwaves at 2.45 GHz. In one such case, microwaves were used to accelerate the rate of an enzyme-catalyzed reaction (Kidwai et al., 1998). In another case, Milestone, Inc. (Monroe, Conn.) sells microwave-absorbing/heating composites of PTFE and graphite which are designed to be dropped into test tubes to accelerate microwave heating of solutions during chemical syntheses. However, in these cases the microwaves are not directed to heat a surface, but used to heat the bulk solution.
In another application, microwaves have been used to heat the bulk solvent during solid-phase combinatorial chemistry (Kappe, 2001; Bradley, 2001; Lidstrom et al., 2001). In these cases, conventional resins (polystyrene, for example) function as solid scaffolds for chemistry. The bulk solution was the target of the microwave heating.
In another case, microwaves were used to accelerate a chromogenic reaction between noble metals and chromogenic reagents. This analytical reaction was performed in solution by flow injection analysis (FIA) (Jin et al., 1999). The reaction depended on bulk solvent heating rather than targeted dielectric material heating.
In yet another case, microwaves were used to enhance the solution phase formation of a fluorescent complex of aluminum (Kubrakova, 2000). The fluorescence intensity could be used to measure aluminum ions in solution. Again, the reaction depended on bulk heating of solvent.
In yet other cases, microwave heating has been used in biochemistry applications. In one instance, microwave heating-assisted protein staining (Nesatyy et al., 2002). In another instance (Boon & Kok, 1989), microwave heating was used to accelerate enzyme-linked immunosorbent assays (ELISAs). In none of these was microwave heating directed to a solid surface, but rather microwave heating was applied to heat a bulk aqueous target.
Natural and Man-Made Enzymes
Nature uses specifically folded proteins called enzymes to catalyze specific reactions necessary for the function of a living organism. Nature also uses non-catalytic proteins, such as receptors and antibodies to affect other biological processes. Both catalytic and non-catalytic proteins have remarkable pockets on their surfaces that bind to the appropriate molecule with exquisite specificity. In the case of enzymes, when the appropriate molecule is bound in the binding pocket (called an “active site”), a chemical reaction takes place that converts the molecule (substrate) into a chemically different molecule (product). The reaction product dissociates from the active site, allowing the (unaltered) enzyme to bind and catalyze another reaction “turnover”.
Protein-based enzymes, receptors, and antibodies are often used in industry, medicine, and diagnostics as reagents. For example, antibodies are used as therapeutic agents for various diseases including cancer and rheumatoid arthritis. Enzymes are used to “fade” denim blue jeans and to process high fructose corn syrup. Antibodies and enzymes are used in immunoassays in medical diagnostics. Despite the widespread use of naturally occurring antibodies and enzymes, many laboratories have sought to create artificial antibodies, receptors, and enzymes. One drawback of the use of natural proteins, or modified natural proteins, for practical purposes, is that proteins are not particularly stable molecules. Artificial reagents would have greater stability to non-physiological temperatures, pH values, non-aqueous solvents, and salt concentrations. Also, natural proteins are susceptible to degradation by contaminating enzymes called proteases that hydrolytically cleave and inactivate other proteins. In addition, even under ideal storage conditions (cold storage in a suitable buffer) the shelf life of proteins can be very short. Finally, in many cases a binding or catalytic reagent is desired for which there is no known natural antibody or enzyme. For example, an antibody may be desired that binds a very small molecule such as methanol or an enzyme may be desired that carries out a chosen stereospecific reaction during preparation of a fine chemical.
Because of the above-stated drawbacks of natural proteins, many laboratories have developed non-protein bio-mimetic compounds that function in the same way as antibodies or enzymes. A wide range of classes of chemical structures has been shown to be useful as artificial proteins. In all cases, the artificial biomolecules have binding pockets that specifically bind to a molecule of choice. These include, but are not limited to; molecularly imprinted polymers (Dai, et al., 1999; Dickert & Thierer, 1996; Leonhardt & Mosbach, 1987), chiral ligands (Maugh 1983a), cavitands (Maugh 1983b, Breslow et al., 1983) and zeolites, and other low molecular weight organic synthetic receptors (Borchart & Clark, 1994). In addition, natural proteins are often sought out or modified to have enhanced stability (thermal or other)(Maugh, 1984).
Individuals who have made artificial enzymes or antibodies/receptors have never reported the possibility that directed microwave energy could be used to promote the rate of a biospecific chemical reaction.
The present invention combines salient features of these two previously unassociated fields. By combining certain aspects of these fields it has been discovered that the rate of chemical reactions can be accelerated by the energy of microwave radiation and with the exquisite regio- and stereo-specificity of natural enzymes.
The present invention reveals a novel means of using microwave energy to specifically accelerate chosen chemical reactions. The reaction specificity comes from the fact that the microwaves are directed to lossy (see definitions below) materials that contain specific binding sites for the desired reactant. The invention describes new uses of microwave radiation. It has never before been disclosed how to direct dielectric heat to accelerate the reaction of a specific molecule in a mixture of similar and/or dissimilar molecules. In this way, the invention describes a new form of artificial enzyme. The results are obtained by using a dielectric material that has substantially better heating properties than water at the chosen microwave emission frequency. A specific reactant-binding molecule is in association with the preferentially heated dielectric material, causing the enhanced reaction of bound reactant.
The present invention also discloses how microwaves can further be used to prepare the surface in advance of such reactions. As described above, natural and artificial enzymes require a specific binding interaction between at least two chemical moieties and an energetic impetus for a reaction to occur (microwave energy in this case). Microwave-facilitated surface preparation includes but is not limited to accelerating the binding of a biomolecule such as a protein or DNA to the surface or accelerating the synthesis of a peptide or other ligand or substrate.